Technical Field
[0001] The present invention relates to a new plate for mass spectrometry that enables proteome
analysis in large amounts, with quickness, and at high sensitivity, a method of preparing
the same, and use thereof.
Background Art
[0002] Owing to the advances in fundamental research into drug innovation, including molecular
biology and genomics (genome science), the circumstances of drug innovation have changed
dramatically over these several years, and new approaches to drug innovation, represented
by genome drug innovation, are being developed.
[0003] However, it remains impossible to predict protein functions (physiological actions)
from the nucleotide sequences clarified by genomics; there is a post-genome need for
the establishment of techniques that link genetic information to new drugs. As such,
proteomics (protein analysis science) is drawing attention, which aims at isolating
and identifying all proteins (reportedly more than 100,000 kinds) translated from
the aforementioned nucleotide sequences, and assigning their functions.
[0004] Although proteome means all proteins that constitute a single organism in a narrow
sense, it sometimes means, in a broad sense, all proteins contained in a histologically
or anatomically specified portion of an organism, like all proteins in the cell fluid
of a particular cell, all proteins contained in serum, all proteins contained in a
particular tissue, and the like. In the present specification, this term is used in
the broad sense, but it is evident that the complete assembly of proteomes in the
broad sense is the proteome in the narrow sense.
[0005] In proteome analysis, a method combining two-dimensional electrophoresis and mass
spectrometry has conventionally been used commonly, but the following problems remain
unresolved. That is, in the conventional method, the migration gel must be divided
into fractions, from each of which proteins must be extracted with a special solution,
before the sample is subjected to mass spectrometry after migration. Due to the long
time taken and the requirement of painstaking operation in multiple stages, measuring
time shortening, apparatus size reduction, screening of a large number of analytes,
and whole-apparatus automation have been extremely difficult.
[0006] Furthermore, there is another problem of what is called "low-abundance proteins".
In yeast, for example, only 100 genes produce 50% of the total protein weight of the
yeast. This means that the remaining 50% proteins are products of thousands of genes.
A large amount of low-abundance proteins contains a large amount of proteins that
are most important to the body, such as regulatory proteins and signal transmission
proteins, including receptors. However, the situation with the conventional method
is such that the large number and trace amount of protein samples separated by electrophoresis
cannot be recovered.
[0007] In an attempt to overcome these limitations in the combined technology of electrophoresis
and mass spectrometry, and to clarify protein-protein interactions, a variety of efforts
are now being made. For example, the isotope-coded affinity tag method (ICAT: isotope-coded
affinity tag;
Nat. Biotech., 17: 994-999 (1999)), the two-hybrid system in yeast, BIA-MS-MS, the protein
array method (solution, chip;
Trends Biotechnol., 19: S34-39 (2001)), peptidomics by LC-MS-MS, and the like are available. In particular,
as examples of the protein array method-, the solid phase protein array method (chip
method;
Curr. Opin. Biotechnol., 12: 65-69 (2001)), the liquid phase array method (fluorescence-encoded beads;
Clin. Chem., 43: 1749-1756 (1997);
Nat. Biotech. , 19: 631-635 (2001); barcoded nanoparticles;
Trends Biotechnol. (2001), ibidem), wherein information has been incorporated in nano-particles, and
the like can be mentioned.
[0008] Meantime, the present inventors have proposed a method of proteome analysis that
enables simultaneous analysis of membrane proteins and compounds capable of interacting
therewith by grouping them (WO 02/56026).
[0009] Aside from such improvements in measuring methods (systems), there are some reports
of attempts for improvements in the field of protein mass spectrometry, though they
are not intended for proteome analysis, or are technically difficult to apply to proteome
analysis. Methods have been reported, which comprise blotting proteins separated by
electrophoresis to a polyvinylidene difluoride (PVDF) membrane, and taking measurements
with the membrane immobilized to a stainless steel plate for MALDI type mass spectrometry
using a double-coated tape (
Electrophoresis, 17: 954-961 (1996)), a frame (
Anal. Chem., 69: 2888-2892 (1997)) or grease (
Anal. Chem., 71: 4800-4807 (1999);
Anal. Chem., 71: 4981-4988 (1999); WO 00/45168). These methods are faulty in that not only the procedures
are painstaking due to immobilization of the PVDF membrane to the plate for mass spectrometry
in the midst of measurement, but also the background is high and the relative peak
intensity is extremely low, so that they are unsuitable to proteome analysis, which
requires high detection sensitivity.
[0010] Also, a method comprising blotting a gel after electrophoresis directly to a matrix-coated
plate for mass spectrometry and conducting mass spectrometry, and a method comprising
electrically blotting a protein to a PVDF membrane for blotting, and then diffusion-blotting
the protein to a matrix-coated plate for mass spectrometry, have been reported (US
5,595,636). However, in these methods, when electrical blotting is used, there is
concern that the matrix dissolves in the blotting buffer during blotting and measurements
themselves become impossible. On the other hand, when diffusion blotting is used,
the blotting efficiency is low so that the detection sensitivity is insufficient particularly
to blotting low-abundance proteins to a plate for mass spectrometry.
[0011] Furthermore, a method based on an improvement of the above-described method has been
reported, which comprises applying a mixture of nitrocellulose (a membrane component
for blotting use) and a matrix to a plate for mass spectrometry as (GB 2312782 A).
However, even this method cannot be said to have completely resolved the problems,
in the case of electrical blotting or diffusion blotting.
[0012] As plates for mass spectrometry, commonly used aluminum or stainless steel plates,
as well as improvements thereof, such as plates for mass spectrometry coated with
silica, or having a hydrophobic group added thereto, are commercially available (manufactured
by Ciphergen, WO 94/28418). However, even these technologies and products do not always
meet the research and development needs for conducting proteome analysis in large
amounts, with quickness, and at high sensitivity.
[0013] An object of the present invention is to introduce a new technology to the conventional
method of proteome analysis based on a combination of electrophoresis and mass spectrometry.
Specifically, the object of the present invention is to provide a technology that
enables the conduct of proteome analysis in large amounts, with quickness, and at
high sensitivity.
Disclosure of the Invention
[0014] The present invention is based on the discovery that a plate for mass spectrometry
prepared by coating a substrate with PVDF to form a thin layer of PVDF on the substrate
offers dramatically improved mass spectrometry detection sensitivity and accuracy
compared to those of one wherein PVDF is immobilized on a substrate in the form of
a membrane. Also, because such a plate for mass spectrometry does not contain a matrix
at the time of blotting, it is additionally advantageous in that the matrix does not
dissolve even in electrical blotting.
[0015] Accordingly, the present invention relates to:
1) a plate for mass spectrometry comprising a support and a PVDF-containing coating
thereon;
2) a method of preparing a plate for mass spectrometry, which comprises coating the
support surface with PVDF; and
3) a method of identifying an analyte, which comprises subjecting an analyte-containing
sample to gel electrophoresis to separate the analyte, blotting the separated analyte
to a plate for mass spectrometry obtained by the method of 1) above or 2) above, and
subjecting the blotted analyte to mass spectrometry.
[0016] Further objectives and features of the present invention and the effect of the present
invention will be evident through the following detailed description of the invention.
Brief Description of the Drawings
[0017]
Fig. 1 shows the extent of electrical blotting from the migration gel to the plate
for mass spectrometry. A indicates the migration gel (before blotting), B indicates
the migration gel (after blotting), and C indicates the plate for mass spectrometry
of the present invention (after blotting). The numerical symbols on the left end show
the molecular weights of the proteins corresponding to the bands. It is shown that
nearly all portions of the proteins in the gel migrate to the plate for mass spectrometry
due to electrical blotting in the case of some proteins.
Fig. 2 shows the positional relationship between a migration gel and a plate for mass
spectrometry (Fig. 2A) and differences in the quantity of laser received by the migrated
protein on the plate (Fig. 2B). In the figures, 1, 2 and 3 indicate a plate for mass
spectrometry, a migration gel, and a protein, respectively. Also, A, B and C indicate
maximum light quantity, small light quantity, and zero light quantity, respectively.
Fig. 3 shows the relative intensities of mass spectrometry peaks obtained using the
plate for mass spectrometry of the present invention (Fig. 3A), an aluminum plate
(Fig. 3B), and a ProteinChip array (H4, Fig. 3G).
Fig. 4 shows the mass spectrometry spectra (peak images) of SCUPA and HSA obtained
using the plate for mass spectrometry of the present invention or an aluminum plate.
The horizontal axis indicates molecular weight, and the vertical axis indicates relative
intensity. Figs. 4A and B show the results for SCUPA, and Figs. 4C and D for HSA.
Also, Figs. 4A and C show the case using the plate for mass spectrometry of the present
invention, and Figs. 4B and D show the case using the aluminum plate.
Fig. 5 shows the results of an analysis of the protein-protein interaction between
a protein (SCUPA) electrically blotted using the plate for mass spectrometry of the
present invention and an antibody that specifically reacts thereto (anti-SCUPA antibody)
by mass spectrometry. The horizontal axis indicates molecular weight, and the vertical
axis indicates relative intensity. The numerical symbols in the Figure indicate the
molecular weights of the peaks. Fig. 5A shows the interaction between the separated
and blotted SCUPA and the anti-SCUPA antibody. Also, Fig. 5B shows the results obtained
using the anti-SCUPA antibody alone.
Fig. 6 shows mass spectrometry spectra obtained when electrophoresis-separated proteins
were blotted to a PVDF membrane. The horizontal axis indicates molecular weight, and
the vertical axis indicates relative intensity. The numerical symbols in the Figure
indicate the molecular weights of the peaks. Fig. 6A shows the results for SCUPA,
and Fig. 6B for HSA.
Best Modes For Embodying The Invention
Plate for mass spectrometry
[0018] The plate for mass spectrometry of the present invention has a coating (thin layer)
containing polyvinylidene difluoride (PVDF) on a support (basic structure). The material
for the support is not subject to limitation, as long as it is one in common use in
plates for mass spectrometry. As examples, insulators (glass, ceramics, plastics/resins
and the like), metals (aluminum, stainless steel and the like), electroconductive
polymers, complexes thereof and the like can be mentioned. Preferably, an aluminum
plate is used.
[0019] The shape of the plate for mass spectrometry of the present invention is designed
to fit particularly to the sample inlet of the mass spectrometer used. For example,
a cluster type plate for mass spectrometry previously notched to enable easy separation
of individual segments so that each fits to the sample inlet of the mass analyze after
a protein is blotted to an assembly type plate for mass spectrometry adapted to the
size of the gel after electrophoresis, and the like can be mentioned, which, however,
are not to be construed as limiting the invention.
[0020] In the present invention, "PVDF-containing coating" refers to a thin layer formed
by depositing PVDF molecules in dispersion on the support, rather than to a layer
prepared by overlaying a previously molded structure on the support as with a conventionally
known PVDF membrane. Although the mode of PVDF deposition is not subject to limitation,
the means mentioned as examples in the method for preparing a plate for mass spectrometry
which is described below are preferably used.
[0021] The thickness of the thin layer can be appropriately chosen, as long as it does not
adversely affect protein blotting efficiency and mass spectrometry measuring sensitivity
and the like, and is, for example, about 0.1 to about 1000 µm, preferably about 1
to about 300 µm.
A method for preparing a plate for mass spectrometry
[0022] The plate for mass spectrometry of the present invention is prepared by coating the
support surface with PVDF. As preferable examples of the means of coating, painting,
spraying, vapor deposition, immersion, printing, sputtering and the like can be mentioned.
[0023] In the case of "painting", PVDF dissolved in an appropriate solvent, for example,
an organic solvent such as dimethyl formamide (DMF), at an appropriate concentration
(for example, about 1 to about 100 mg/mL) (hereinafter referred to as "PVDF-containing
solution"), can be painted to the support (basic structure, substrate) using an appropriate
tool such as a brush.
[0024] In the case of "spraying", a PVDF-containing solution prepared in the same manner
as above may be sprayed from a sprayer so that PVDF is uniformly deposited on the
support.
[0025] In the case of "vapor deposition", a thin layer of PVDF can be formed on the support
surface by heating and vaporizing PVDF (may be solid or solution) in a vacuum chamber
containing the support using an ordinary vacuum vapor deposition apparatus for preparation
of organic thin membranes.
[0026] In the case of "immersion", the support may be immersed in a PVDF-containing solution
prepared in the same manner as above.
[0027] In the case of "printing", various commonly usable printing technologies can be appropriately
chosen and utilized according to the material of the support; for example, screen
printing and the like can be preferably used.
[0028] In the case of "sputtering", a thin layer can be formed, for example, by applying
a direct current high voltage between the support and PVDF while an inert gas (e.g.,
Ar gas and the like) is introduced into a vacuum, and colliding the ionized gas to
the PVDF to allow the sputtered PVDF molecules to deposit on the support.
[0029] A coating may be applied to all faces of the support, and may be applied only to
one face subjected to mass spectrometry.
[0030] PVDF can be used appropriately in a preferred form according to the coating means;
for example, PVDF can be applied to the support in the form of a PVDF-containing solution,
PVDF-containing vapor, PVDF solid molecules and the like, and it is preferably applied
in the form of a PVDF-containing solution. "Apply" means that PVDF is brought into
contact with the support so that it remains and accumulates on the support after contact.
The amount applied is not subject to limitation; as an example of the amount of PVDF,
about 1 to about 100 µg/cm
2 can be mentioned. After application, the solvent is removed by spontaneous drying,
vacuum drying and the like.
[0031] The support (basic structure, substrate) in the plate for mass spectrometry of the
present invention may have the surface thereof previously modified (processed) by
an appropriate physical or chemical technique before being coated with PVDF. Specifically,
techniques such as plate surface polishing, abrasion, acid treatment, alkali treatment,
and glass treatment (tetramethoxysilane and the like) can be mentioned as examples.
[0032] The plate for mass spectrometry of the present invention is excellent in stability.
That is, the plate for mass spectrometry of the present invention is characterized
in that the adhesive surface does not peel even under such conditions as immersion
in an aqueous solution having a pH of 2 to 10, or containing various salts, a solvent
such as methanol or acetonitrile, or a mixed solvent thereof, electrical loading,
repeated cycles of wetting and drying, and a highly vacuum state.
Use of the plate
A. Identification of proteins and the like
[0033] Using the plate for mass spectrometry of the present invention, various compounds,
including proteins, can be identified. That is, by subjecting a sample containing
an analyte (e.g.: protein, nucleic acid, oligonucleotide, saccharide, oligosaccharide,
cell membrane receptor agonist or antagonist, toxin, virus epitope, hormone, peptide,
enzyme, enzyme substrate or inhibitor, cofactor, drug, lectin, antibody and the like)
to an electrophoresis (e.g.: polyacrylamide gel electrophoresis) to separate the analyte,
blotting the separated analyte to the plate for mass spectrometry of the present invention,
and analyzing the blotted analyte by mass spectrometry, the analyte is identified
(from information on the molecular weight thereof).
Analyte-containing sample
[0034] The analyte-containing sample may be prepared from a (biological) material using
any known technique. Here, as examples of the (biological) material, cells or tissues
of optionally chosen organisms such as animals, plants and microorganisms, or extracellular
fluids (for example, blood, plasma, urine, bone marrow fluid, ascites fluid and the
like), intracellular fluids, fluids in cell small granules, and the like can be mentioned.
Also, cell culture broths, culture broths obtained by gene recombination, and the
like are also included.
[0035] To prepare an analyte-containing sample subjected to electrophoresis, a known method
is used. For example, in the case of a protein-containing sample, a soluble fraction
can be obtained by collecting a centrifugal supernatant after target cells are homogenized
in an appropriate buffer in the presence of various protease inhibitors, or after
the cells are suspended using a cell disruption apparatus such as Polytron, or after
the cells are disrupted by hypo-osmotic shock, or after the cell membranes are disrupted
by sonication. Also, the obtained precipitate (insoluble) fraction can be used as
after being solubilized using a surfactant or a protein denaturant.
Electrophoresis
[0036] This is a step wherein the analyte in the sample is separated (developed on gel)
by electrophoresis. The electrophoresis apparatus used can be a known one. A commercial
product may be used. According to the purpose, any of one-dimensional gel electrophoresis
and two-dimensional gel electrophoresis can be used. In two-dimensional gel electrophoresis,
one-dimensional migration is based on separation according to analyte isoelectric
point, and two-dimensional migration on separation according to analyte molecular
weight. The size of the gel used for electrophoresis is not subject to limitation.
Although 10 cm × 10 cm is a commonly used size, 20 cm × 20 cm or other sizes can be
used if necessary. Also, although the material of the gel is basically polyacrylamide,
utilization of other media such as agarose gel and cellulose acetate membrane is also
possible depending on the purpose. For the concentration of the gel, both a uniform
concentration and a gradient concentration can be used.
Blotting
[0037] This is a step wherein the analyte separated by gel electrophoresis is transferred
from the gel to the plate for mass spectrometry of the present invention. As the blotting
apparatus, a known one can be used. A commercial product may be used. The method of
blotting is known per se. The analyte developed on the gel after migration is transferred
to the plate for mass spectrometry by various methods (diffusion, electrical force
and others). This step is generally called blotting. Diffusion blotting, electrical
blotting and the like can be mentioned. Electrical blotting is particularly preferable.
[0038] As the buffer used at the time of electrical blotting, it is preferable to use one
of a pH of 7 to 9 and of a low-salt concentration. Specifically, a Tris buffer, a
phosphate buffer, a borate buffer, an acetate buffer and the like can be mentioned
as examples. As the Tris buffer, Tris/glycine/methanol buffer, SDS-Tris-Tricine buffer
and the like can be mentioned; as the phosphate buffer, ACN/NaCl/isotonic phosphate
buffer, sodium phosphate/ACN and the like can be mentioned; as the borate buffer,
sodium borate-hydrochloride buffer, Tris-borate salt/EDTA, borate salt/ACN and the
like can be mentioned; as the acetate buffer, Tris-acetate salt/EDTA and the like
can be mentioned. Preferably, the buffer is Tris/glycine/methanol buffer or sodium
borate-hydrochloride buffer. As examples of the composition of the Tris/glycine/methanol
buffer, 10-15 mM Tris, 70-120 mM glycine, 7-13% methanol or so can be mentioned. As
examples of the composition of the sodium borate-hydrochloride buffer, 5-20 mM sodium
borate or so can be mentioned.
[0039] Also, after the blotting, a reagent called matrix can be added to absorb laser, and
to promote the ionization of analyte molecules through energy transfer, so that the
subsequent mass spectrometry (by the MALDI method) is effected. As the matrix, one
known in the field of mass spectrometry can be used. For example, sinapinic acid (SPA
(= 3,5-dimethoxy-4-hydoroxycinammic acid)), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic
acid (DHB), α-cyano-4-hydroxycinammic acid (CHCA) and the like can be mentioned, which,
however, are not to be construed as limiting the matrix. Preferably, the matrix is
DHB or CHCA. In the case of CHCA, it is preferable to add one of at least 21% saturation
concentration. Specifically, those of 21 to 100% saturation concentration, preferably
those of 40 to 100% saturation concentration, and particularly preferably those of
50 to 100% saturation concentration can be mentioned.
Mass spectrometry
[0040] This is a step wherein the analyte is identified (from information on molecular weight)
by analyzing the analyte blotted to the plate for mass spectrometry of the present
invention by mass spectrometry. The mass spectrometer is an apparatus that measures
and detects the molecular weight of a substance by ionizing a gaseous sample, thereafter
bringing molecules or molecule fragments thereof into an electromagnetic field, separating
the substance by mass number/charge number based on the transfer status thereof, and
determining the spectrum of the substance. It is possible to use mass spectrometers
based on the principles of the MALDI-TOFMS method, which is a combination of matrix-aided
laser deionization (MALDI), comprising mixing and drying a sample and a laser-absorbing
matrix to cause crystallization, subjecting the crystal to ionization by energy transfer
from the matrix and instantaneous heating by laser irradiation, and introducing the
ionized analyte into a vacuum, and time-of-flight mass spectrometry (TOFMS), comprising
analyzing mass number by sample molecule ion time-of-flight differences due to initial
acceleration; the method comprising placing a single analyte on one droplet and electrically
ionizing the analyte directly from the liquid; the nano-electrospray mass spectrometry
(nano-ESMS) method, comprising electrically spraying a sample solution into the atmosphere
to vaporize individual analyte polyvalent ions in an unfolded state, and the like.
[0041] The method of subjecting an analyte on the plate for mass spectrometry of the present
invention to mass spectrometry is known per se.
[0042] Also, for fully automating proteome analysis according to the present invention,
it is possible to analyze all analytes developed one-dimensionally or two-dimensionally
by electrophoresis by moving the laser exit or, conversely, the stand with the plate
for mass spectrometry on, toward one-dimensional or two-dimensional directions, and
performing continuous full-surface scans (in the intermittent scan method, there remain
analytes not irradiated with laser, that is, not analyzed). Combining this method
with the intermittent scan method makes it possible to mount 96 kinds of samples dispensed
to a 96-well plate as a whole to a plate for mass spectrometry (96 kinds of samples
arranged at constant intervals in a rectangular chip), and subject the samples as
is to mass spectrometry.
B. Identification of a group of analytes
[0043] According to the present invention, it is possible to analyze an assembly of a plurality
of kinds of analytes (a group of analytes) at one time. That is, the analyte-containing
sample prepared from the above-described various biological materials usually contains
many kinds and a wide variety of analytes. By separating such a group of analytes
by one-dimensional or two-dimensional gel electrophoresis, and blotting the group
of analytes separated and developed on the gel to, for example, a plate for mass spectrometry
of the present invention of a size that fits to the migration gel (preferably a cluster
type plate for mass spectrometry having perforated lines made previously to enable
easy separation of each piece so that it fits to the sample inlet of the mass spectrometer
after blotting), it is possible to fractionate all, as a rule, analytes contained
in the analyte-containing sample (proteomes if the analytes are proteins) to respective
positions and blotting them to the plate for mass spectrometry. By measuring these
a group of analytes using the above-described continuous scan type mass spectrometer,
the blotted a group of analytes can be identified at one time.
C. Identification of analyte complex
[0044] According to the present invention, the linkage of an analyte and one that has affinity
for (interacts with) the analyte can be analyzed at one time. That is, by separating
the analyte by electrophoresis, blotting the separated analyte to the plate for mass
spectrometry of the present invention, thereafter adding a sample containing a compound
that has affinity for (interacts with) the analyte (for example, peptide, protein,
nucleic acid, non-peptide compound, synthetic compound, fermentation product, cell
extract, plant extract, animal tissue extract,cell membrane fraction, organelle membrane
fraction and the like) to form a complex on the plate, and subjecting the formed complex
to mass spectrometry, the blotted analyte, a compound that has affinity (interacts
with) the analyte, and/or a complex thereof is identified (from information on molecular
weight).
[0045] To describe the present invention in more detail, Examples are given below, which
examples, however, are not to be construed as limiting the present invention.
Example 1: Preparation of plate for mass spectrometry
[0046] An aluminum basic structure (aluminum plate) prepared to fit to the inlet of the
plate for mass spectrometry of the ProteinChip System (manufactured by Ciphergen)
was immersed in 1% tetramethoxysilane/1% acetic acid solution and air-dried, after
which it was burned (130°C, 3 hours). To this plate, polyvinylidene difluoride (PVDF)
dissolved in dimethylformamide (DMF) to 10 mg/mL was applied. The plate for mass spectrometry
prepared was tabular and 78 mm × 8 mm × 2 mm in size, and had a white surface.
[0047] This plate for mass spectrometry suffered absolutely no chemical, electrical, mechanical
or physical changes such as cracking, peeling, damage and discoloration, in the subsequent
steps such as pretreatment by immersion in an organic solvent, contact with electrophoresis
gel, electrical blotting in various buffers, and high vacuums during subsequent matrix
application and drying and mass spectrometry.
[0048] Using this plate for mass spectrometry, the following investigation was conducted.
Example 2: Electrical blotting of protein to plate for mass spectrometry
[0049] After a prestained molecular weight marker (manufactured by Bio-Rad) was subjected
to 12% SDS-polyacrylamide gel electrophoresis, the protein on the gel was electrically
blotted to the plate for mass spectrometry prepared in Example 1 at 90 mA for 3 hours
in 12.5 mM Tris/96 mM glycine/10% methanol buffer. As a result, although small amounts
of prestained proteins having molecular weights of 90,000 and 110,000 remained in
the polyacrylamide gel after blotting (Fig. 1B), all proteins were efficiently blotted
to the plate for mass spectrometry of the present invention (Fig. 1C).
Example 3: Mass spectrometry for protein blotted to plate for mass spectrometry
[0050] After a sample (protein mixture) was subjected to 12% SDS-polyacrylamide gel electrophoresis,
the gel was brought into direct contact with the plate for mass spectrometry of Example
1, the protein separated by electrophoresis was electrically blotted to the plate
for mass spectrometry prepared in Example 1, and this plate for mass spectrometry
was immediately placed in a mass spectrometer and measured. Using 2,5-dihydroxybenzoic
acid (DHB; 75 mg/mL ethanol solution) as the matrix, and using the ProteinChip System
(described above) as the measuring apparatus, measurements were taken under the conditions
of a detector voltage of 1800 V, a detector sensitivity of 8, and a laser intensity
of 280. For mass calibration, external calibration was conducted using myoglobin (derived
from equine muscle), GAPDH (derived from rabbit), and albumin (derived from bovine
serum). In this experiment, two kinds of proteins, namely a single chain urinary plasminogen
activator (SCUPA) and human serum albumin (HSA), were used as the protein mixture,
and 4 µg of the protein mixture was twice electrophoresed tandem to 12% SDS-polyacrylamide
gel under reducing conditions. That is, an initial sample was added, and electrophoresis
was performed at 30 mA for 40 minutes, after which the electricity was broken, the
same sample was again added to the same lane, and electrophoresis was further performed
at 30 mA for 23 minutes. After migration, the gel was cut for each migration lane,
and further cut at a position 39 mm from the addition site on the upper face of the
gel, after which the two pieces of the gel were placed with their upper faces in contact
with each other, and thereafter brought into contact with the plate for mass spectrometry
(Fig. 2A). At this time, the migration samples were arranged in the order of SCUPA,
HSA, SCUPA, HSA, HSA, SCUPA, HSA and SCUPA, and four bands of each protein, eight
bands in total, were blotted to the plate for mass spectrometry.
[0051] Because the mass spectrometer used irradiates laser to a single plate for mass spectrometry
not continuously but at 24 equally spaced points, there occurs a case of no-exposure
to laser, a case of partial exposure, and a case of full exposure, depending on the
position of the blotted protein on the plate for mass spectrometry (Fig. 2B). To increase
the probability of the greatest exposure to laser and hence the highest peak intensity
with this limitation in the analytical apparatus, a mode was designed to blot each
model protein to four different positions on the plate for mass spectrometry by the
technique described above. As a result, even when proteins of theoretically the same
mass were blotted, the heights of the peaks obtained from mass spectrometry were variable
(Fig. 3). Hence, in this experiment, a discussion was made assuming the peak of the
largest relative intensity out of the plurality of peaks obtained to be the value
closest to the true value of complete exposure to the laser.
[0052] The plate for mass spectrometry was previously immersed in methanol and equilibrated
with a blotting buffer (10mM sodium borate buffer, pH 8.0), after which it was subjected
to electric blotting at 90 mA for 2 hours. Subsequently, mass spectrometry was conducted
and relative peak intensity was measured. For reference controls, other experiments
were conducted in the same manner but using an ordinary non-PVDF-coated aluminum plate
and an aluminum plate having a hexadecyl group introduced thereto (ProteinChip array,
manufactured by Ciphergen, H4). The results are shown in Fig. 3 and Fig. 4.
[0053] In the case of SCUPA, the relative peak intensity for the plate for mass spectrometry
of the present invention (Fig. 3A) was 12.31, a level 14 times as high as the value
of the ordinary aluminum plate (Fig. 3B) (0.87). Making a similar comparison for HSA,
the relative peak intensity for this plate for mass spectrometry was 49 times (6.26:0.129)
as high as the value of the aluminum plate. Also, the plate for mass spectrometry
produced 2 times to 4 times as high relative peak intensity as those from H4 (Fig.
3C). Furthermore, the plate for mass spectrometry of the present invention (Figs.
4A and C) produced more distinct spectra than those from the aluminum plate (Figs.
4B and D). From these results, it was found that by using the plate for mass spectrometry
of the present invention, a plurality of kinds of proteins, after separation by electrophoresis,
could be analyzed by mass spectrometry with quickness and at high sensitivity at one
time.
Example 4: Analysis of interaction between electrically blotted protein and binding
protein after electrophoresis separation
[0054] As an example application of the plate for mass spectrometry of the present invention,
an investigation was conducted on the binding of a protein blotted to a plate for
mass spectrometry and a protein capable of interacting therewith, and on the identification
of the protein complex thereof by subsequent mass spectrometry. As the materials,
SCUPA and an antibody thereof (anti-SCUPA antibody) were used.
[0055] After SCUPA (4 µg) was added to 12% SDS-polyacrylamide gel, electrophoresis was performed
for 1 hour. After migration, the gel was cut, and the SCUPA on the gel was electrically
blotted to the plate for mass spectrometry prepared in Example 1, in the same manner
as Example 3. After the SCUPA-blotted plate for mass spectrometry was blocked, an
antiserum (crudely purified using ammonium sulfate, and containing anti-SCUPA antibody)
was added, and an overnight reaction was carried out. After completion of the reaction,
the plate was washed with PBS buffer, a matrix was added, and mass spectrometry was
conducted. The results are shown in Fig. 5.
[0056] The SCUPA blotted to the plate for mass spectrometry after electrophoresis produced
a peak at 48,616, and the peak of the anti-SCUPA antibody that interacted with the
SCUPA immobilized on the same plate was observed at 145,300 (73,115 assigned to the
divalent ion of the same antibody) (Fig. 5A). Also, in the SCUPA-free gel as the control,
no peaks were observed in this plate for mass spectrometry, despite that similar procedures
such as electrical blotting, blocking, addition of anti-SCUPA antibody, and washing
with PBS were followed (Fig. 5B).
[0057] From the above results, which show that after electrophoresis separation, the SCUPA
blotted to this plate for mass spectrometry retained activity to bind to a protein
capable of interacting therewith (anti-SCUPA antibody), and that the molecular weights
of both the blotted protein and the protein interacting therewith were actually determined
at one time, resulting in the identification of both molecular species, it was demonstrated
that detection of the protein-protein complex on this plate for mass spectrometry
and identification of the complex produced on this plate for mass spectrometry are
possible.
Comparative Example 1: Blotting to PVDF membrane and mass spectrometry
[0058] On 12% SDS-polyacrylamide gel, a mixture of 4 µg of each of two kinds of proteins,
namely SCUPA and HSA, was twice electrophoresed tandem under reducing conditions.
An initial sample was added, and electrophoresis was performed at 30 mA for 40 minutes,
after which the electricity was broken, the same sample was again added to the same
lane, and electrophoresis was further performed at 30 mA for 23 minutes. After migration,
the gel was cut for each migration lane, and further cut at a position 39 mm from
the addition site on the upper face of the gel; as illustrated in Fig. 2A, the two
pieces of the gel were placed with their upper faces in contact with each other, and
a 78 mm × 8 mm cut PVDF membrane was placed on the gels, and electrical blotting was
conducted in 10 mM sodium borate buffer (pH 8.0) at 90 mA for 2 hours. After completion
of blotting, the PVDF membrane was washed with PBS, rinsed with distilled water, dried,
and applied to an aluminum plate using a transparent a double-coated tape (manufactured
by Sumitomo 3M Ltd.). A matrix (DHB) was added thereto, and mass spectrometry was
conducted using the ProteinChip System (described above).
[0059] As a result, the SCUPA blotted to the PVDF membrane produced a peak at 50,096.9,
and the HSA at 67,394.5; however, the peaks were difficult to identify because the
relative peak intensities were extremely low, and also because the S/N ratios were
low (Fig. 6). Also, it was found that in the case of a plate for mass spectrometry
(the same applies to the plate for mass spectrometry of the present invention), 2
to 3 minutes were taken to reach a high vacuum state, immediately after which measurements
were possible, whereas in the case of a PVDF membrane, as long as 45 minutes were
taken to reach a vacuum state, resulting in an excess load on the mass spectrometer
used, and the mass spectrometer did not endure frequent use.
Example 5: Various proteome analyses using the plate for mass spectrometry of the
present invention
[0060] Using various cell or tissue extraction samples, the performance of the plate for
mass spectrometry of the present invention was confirmed in a range of relatively
low molecular weights (molecular weight 1,000 to 20,000). In the following experiment,
the sample was applied to 16% SDS-polyacrylamide gel (in Tris-Tricine buffer) and
electrophoresed for 90 minutes, after which the analyte was electrically blotted from
the gel to the plate for mass spectrometry of the present invention in 10 mM sodium
borate buffer (adjusted to pH 8.0 with hydrochloric acid) for 1 to 2 hours. After
completion of blotting, the plate was rinsed, the matrix was spotted, and mass spectrometry
was conducted.
(1) A swine cerebellum was homogenized with IN acetic acid at 4°C and centrifuged
at 4°C and 3,000 rpm for 30 minutes, and the supernatant was recovered. Acetonitrile
was added to obtain a final concentration of 10%, and the supernatant was applied
to a column for reversed-phase chromatography. The column was washed with 0.1% trifluoroacetic
acid (TFA) containing 10% acetonitrile, and eluted with 0. 1% TFA containing 60% acetonitrile.
The eluate was freeze-dried and used as the swine cerebellum extract. After this extract
was separated by SDS-PAGE, it was electrically blotted to the plate for mass spectrometry
of the present invention. Using 0.5% TFA/50% ACN containing saturated α-cyano-4-hydroxycinnamic
acid (CHCA) as the matrix, mass spectrometry was conducted. As a result, peaks were
detectable in the molecular weight range of 3,000 to 20,000.
This was compared with cases wherein DHB (150 mg/mL ethanol) or saturated SPA (in
0.5% TFA/50% ACN) was used in place of saturated CHCA as the matrix. In the case of
DHB, no distinct peaks were observed in the molecular weight range of 3,000 to 20,000.
In the case of saturated SPA, only several peaks were observed in the same molecular
weight range. In contrast, in the case of saturated CHCA, a large number of peaks
were observed in the molecular weight range of 2,000 to 10,000 and in the molecular
weight range of 5,000 to 20,000. From these results, it was shown that CHCA was more
preferable for identification of proteins in relatively low molecular weight ranges.
(2) Mass spectrometry of swine cerebellar extract was conducted in the same manner
as (1) above except that 50% saturated CHCA was used as the matrix. As a result, peaks
were detectable in the molecular weight range of 1,000 or more. In particular, excellent
performance was shown in detection of peaks in the molecular weight range of 3,000
to 20,000.
This was compared with a cases using 10 or 20% saturated CHCA in place of 50% saturated
CHCA, in terms of the extent of peak appearance in mass spectrometry in the molecular
weight range of 1,000 to 10,000. In the case of 10% saturated CHCA, several peaks
were observed. In the case of 20% saturated CHCA, the number of peaks was slightly
larger than that for 10% saturated CHCA. In contrast, a large number of peaks were
observed in the case of 50% saturated CHCA.
(3) U937 cells (1 ×10
5 to 2 × 10
6 cells/ml) were cultured in a RPMI 1640 medium containing 10% FCS in the presence
of 100 ng/ml phorbol 12-myristic acid ester 13-acetic acid salt (PMA) for 48 hours.
The same experiment, but in the absence of PMA, was conducted for control. After completion
of cultivation, the cells were washed with phosphate-buffered saline (PBS), and cell
pellets were prepared. Furthermore, a protein extraction reagent (Novagen Inc.) and
a protease inhibitor were added, and the pellets were allowed to stand at 4°C for
15 minutes, after which they were centrifuged, and the supernatant was recovered and
used as the cell lysate. After this was separated by SDS-PAGE, it was electrically
blotted to the plate for mass spectrometry of the present invention. Subsequently,
mass spectrometry was conducted; in terms of the intensities of the peaks detected
in the molecular weight range of 3,000 to 20,000, the results for the PMA-stimulated
U937 cells were compared with the results for the control U937 cells. As a result,
the following changes in the PMA-stimulated expression of proteins (peak intensity)
were observed (Table 1).
Table 1
Changes in peak (intensity) |
Peak number |
Disappeared |
38 |
Appeared |
11 |
Decreased |
1 |
Increased |
2 |
(4) Profiling of mouse tissues was conducted. Mouse tissues (brain, lung, liver, muscle)
were disrupted by treatment using the Beads Shocker (Yasui Kikai Corporation, Osaka)
at 2,500 rpm for 10 to 30 seconds, TricinePAGE sample buffer (Wako Pure Chemical Industries)
was added, centrifugation was conducted at 12,000 rpm for 5 minutes, and the supernatant
was recovered. After separation by SDS-PAGE, the supernatant was electrically blotted
to the plate for mass spectrometry of the present invention, and mass spectrometry
was conducted. As a result, a tissue-specific peptide (molecular weight 3,000 to 20,000)
was detected in each tissue.
Example 6: Investigation of effects of buffer at the time of electrical blotting
[0061] As the buffer at the time of electrical blotting, a Tris buffer (25 mM Tris/192 mM
glycine/20% methanol, pH 8.3), a phosphate buffer (5% ACN/125 mM NaCl/PBS, pH 7.2),
an acetate buffer (40 mM Tris-acetate salt/1 mM EDTA, pH 8.0), and a borate buffer
(10 mM sodium borate-hydrochloric acid, pH 8.0) were used. As the proteins for mass
spectrometry, HSA and SCUPA were used. Except for these conditions, an experiment
was conducted in accordance with the method of Example 5. As a result, of the buffers
examined, the borate buffer gave the maximum mass spectrometry peak intensity. For
example, the ratio of maximum value of peak intensity for the borate buffer versus
the phosphate buffer was about 21 times for SCUPA and about 4 times for HSA.
Industrial Applicability
[0062] Owing to the use of PVDF as the ligand adsorbent, the plate for mass spectrometry
of the present invention is capable of uniformly adsorb various proteins, is excellent
in blotting efficiency, and enables the normal retention of the three-dimensional
structures and functions of the proteins. The plate for mass spectrometry of the present
invention is also characterized by the absence of non-specific adsorption, the capability
of performing mass spectrometry immediately after blotting, and the like.
[0063] Making use of these features of the plate for mass spectrometry of the present invention,
a protein can be analyzed and identified in large amounts, with quickness, and at
high sensitivity. The protein may be of one kind, or an assembly of a plurality of
kinds (a group of proteins), and even a complex with a compound that has affinity
for (interacts with) the protein can be analyzed and identified likewise (that is,
in large amounts, with quickness, and at high sensitivity).
[0064] Also, significant shortening of operating time, step simplification, apparatus size
reduction, price reduction, automation, and screening of a large number of analytes
are enabled, and large-scale proteome analysis is facilitated. Furthermore, proteome
analysis of physiologically important low-abundance proteins is enabled, thus contributing
to the discovery of biomarkers for diagnostic use and the discovery of seed compounds
for new drug development.
[0065] Therefore, the present invention makes it possible to introduce a new technique to
the conventional proteome analysis, which is a combination of electrophoresis and
mass spectrometry.
[0066] The present application is based on US 10/264,505 filed in the United States and
Japanese Patent Application No. 2002-344710 filed in Japan, the teachings of which
are included in their entirety in the present specification.
[0067] All citations, including the publications and patents mentioned herein, are incorporated
in the specification, by being cited herein, to the same extent that they are individually
shown to be incorporated by reference and all of them are specified herein.
[0068] Although the present invention has been described with emphasis on preferred embodiments,
it is obvious to those skilled in the art that the preferred embodiments may be modified.
The present invention may be embodied by methods other than those described in detail
herein. Therefore, the present invention includes all modifications encompassed in
the spirit and scope of the attached claims.